There is an increasing demand to produce transportation fuels from renewable resources as evidenced through the Energy Independence and Security Act of 2007 (Bingaman, 2007, from the Energy Independence and Security Act of 2007). Biodiesel offers a sustainable alternative to traditional fossil based fuels as it is sourced from a renewable feedstock, with other advantages in that it is cleaner burning than diesel, safer to handle, and promotes longer engine life (Bozbas, 2008, Renew. Sustain. Energy Rev. 12, 542-552). Additionally, biodiesel provides a direct drop-in replacement for petroleum-based diesel fuel and can be used in other applications, for example, boilers (Carraretto et al., 2004, Energy 29:2195-2211). While biofuels (both biodiesel and bioethanol) provide a potentially sustainable fuel source, significant gains towards a favorable energy balance for fuel production would arise from lowering the agricultural inputs required for feedstock production (fertilizers, pesticides, etc.), producing crops on less desirable land, lowering the energy involved with extraction of desirable components and conversion into the fuel, as well as developing a reliable stream of value-added co-products (Hill et al., 2006, Proc. Natl. Acad. Sci. 103:11206-11210).
Life cycle analyses (LCA) of the biodiesel production process from various renewable feedstocks (Yee et al., 2009, App. Energy 86:S189-S196; Bernesson et al., 2004, Biomass and Bioenergy 26:545-559; Brenter et al., 2011, Environ. Sci. Tech. 45:7060-7067; Janulis, 2004, Renew. Energy 29:861-871; Lardon et al., 2009, 43:6475-6481; Gerpen, 2005, Fuel Process. Tech. 86:1097-1107; Kiwjaroun et al., 2009, J. Cleaner Prod. 17:143-153) show that significant energy requirements are involved with the growth of the fuel feedstock as well as lipid extraction and conversion. Several of these LCAs have specifically compared different means for lipid extraction and conversion (Brenter et al., 2011, Environ. Sci. Tech. 45:7060-7067; Lardon et al., 2009, 43:6475-6481; Kiwjaroun et al., 2009, J. Cleaner Prod. 17:143-153). From these studies it can be concluded that compared to current technologies for transesterification, significant benefits can be achieved from more selective and energy efficient processes. One of these studies analyzed biodiesel production from algae and suggested that one-step extraction of triacylglycerides and conversion via transesterification using supercritical methanol (scMeOH) (Patil et al., 2011, Bioresource Tech. 102:118-122) has the greatest potential in terms of minimizing energy requirements.
These gains could be realized since the process does not require dried algae as a starting material, and it combines the two processing steps (extraction and conversion) into a single step, which would minimize the energy needed for solvent production and product separations. While this technology offers energy savings over the base case there is still a question of a positive energy balance mainly due to the high heating requirements associated with producing supercritical methanol.
Another study assessing the transesterification of palm oil analyzed in detail the use of scMeOH for the transesterification of triacylglycerides (Kiwjaroun et al., 2009, J. Cleaner Prod. 17:143-153). This study indicated that while the use of scMeOH provides benefits in terms of improvements to fuel quality, that significant advances must be made in order to decrease the significantly elevated energy needs of producing scMeOH. These high energy requirements may be mediated by lowering the reaction temperature and pressure, as well as by lowering the energy associated with methanol recovery. A critical step towards a more efficient pathway for biodiesel production would be to address the high energy demands of scMeOH transesterification associated with the high critical temperature.
Supercritical transesterification of triacylglyceride feedstocks has been demonstrated using both methanol and ethanol (Pinnarat and Savge, 2008, Ind. Engineer. Chem. Res. 47:6801-6808; Sawangkeaw et al., 2010, J. Supercrit. Fluids 55:1-13). Generally it was found that the most desirable reaction conditions for non-catalytic supercritical transesterification of triacylglyceride feedstocks is between 250-400° C., 19-45 MPa, with approximately a 40:1 methanol to TAG molar ratio for a period of time between 4 and 30 minutes. The use of neat methanol would appear to be the more efficient route, yet it has been determined that methanol and typical triacylglycerides do not form a single phase at temperatures below 225° C. (Hegel et al., 2008, Fluid Phase Equil. 266:31-37; Tang et al., 2006, Fluid Phase Equil. 239:8-11; CerCe et al., 2005, Ind. Engineer Chem. Res. 44:9535-9541). As such, equipment costs and energy demand are significantly elevated for neat methanol systems that must operate in a single fluid phase for reasonable yields. The use of catalysts and/or co-solvents has been explored to moderate these reaction conditions (Cao et al., 2005, Fuel 84:347-351; Demirbas, 2008, Energy Sources, Part A: Recovery, Utilization, and Environmental Effects, 30:1830-1834; Patil et al., 2009, Energy & Fuels 24:746-751). Co-solvents such as propane or hexane (Cao et al., 2005, Fuel 84:347-351) have been added to supercritical methanol in order to form a single fluid phase at less energy intensive conditions. While this strategy is successful from a phase behavior perspective (at mild pressures), it is burdened by flammability issues. Additionally, both the use of neat methanol and methanol/organic co-solvent gives rise to an additional problem in downstream separations of the reaction products. Both fatty acid methyl esters (FAME) and glycerol products are soluble in methanol (Hegel et al., 2008, Fluid Phase Equil. 266:31-37) incurring potentially high separation costs in order to recover the desirable product, FAME, and isolate the undesirable by-product, glycerol. The use of homogeneous catalysts in supercritical transesterification (Patil et al., 2009, Energy & Fuels 24:746-751) may also mediate reaction conditions but has similar challenges in terms downstream separation issues.
Carbon dioxide (CO2) has been used as a co-solvent for transesterification in both supercritical ethanol (Bertoldi et al., 2009, Energy & Fuels 23:5165-*5172 and methanol (Han et al., 2005, Proc. Biochem. 40:3148-3151) with the aim of reducing reaction temperatures and pressures. It was found to be effective at percentages up to 10% CO2, but temperatures still needed to be at least 280° C. in order for the reaction to reach a 98% yield (Han et al., 2005, Proc. Biochem. 40:3148-3151). Two studies explored using methanol in continuous reactors with supercritical CO2 (scCO2) over fixed catalyst beds (Maçaira et al., 2011, Fuel 90:2280-2288; Jackson and King, 1996, J. Amer. Oil Chem. Soc. 73:353-356). The first flowed scCO2 over a bed of lipase finding 98% yield of FAME (Jackson and King, 1996, J. Amer. Oil Chem. Soc. 73:353-356), but high pressures of 17.2 MPa and the expensive catalyst pose potential challenges for scale up and commercialization. The other study used a packed bed of Nafion®-SAC13 in a continuous system consisting of 75:25 CO2:methanol. This study also required high temperatures and pressures (200° C., 25 MPa) (Macaira et al., 2011, Fuel 90:2280-2288). In addition to high pressures, both studies suffered from difficulties pertaining to the downstream separation of glycerol from the product. The use of CO2 in these systems facilitates the solubility of the triacylglyceride and methanol, but the requirement of supercritical conditions may not be necessary in order to benefit from using CO2 (i.e. increased solubility and selectivity as well as decreased mass transfer resistance).
Operating at lower temperatures and pressures in a multi-phase, liquid-vapor system, may allow for similar benefits without such high energy burdens (Beckman, 2003, Environ. Sci. Tech. 37:5289-5296). One other study has assessed the transesterification of rapeseed oil with scCO2 and methanol over sulfonated polymer matrices (Galia et al., 2011, J. Supercrit. Fluid 56:186-193). The authors achieved a maximal 62.4% yield at 140° C. and 11.0 MPa during an 8 h reaction (Methanol/oil 27.7 mol/mol, catalyst loading of 10% w/w substrate). This work undertakes a more fundamental approach to comprehend this complex system consisting of CO2, methanol, substrate, and catalyst and inform the experimental conditions favourable for reaction. Understanding the fluid phase behavior that will contribute to high reaction yields while minimizing the required process energy will be key to efficient transesterification. CO2 has great potential for efficient extraction and production of various fuel and non-fuel products due to its selectivity and flexibility. Efficient process design could not only allow for great energy savings but also support the concept of a biorefinery (Foley et al., 2011, Green Chem. 13:1399-1405) where fuel as well as value-added chemicals can be efficiently produced to increase economic favorability of renewable feedstocks.
Full utilization of biomass for fuels and valuable co-products, analogous to petroleum refining for a wide spectrum of products, has been put forward as a critical research goal. For example, the US DOE Energy Efficiency & Renewable Energy Integrated Biorefinery Program highlights the importance of co-products from energy crops, agricultural residues, and microalgae, to reduce economic and environmental barriers to large-scale fuel production (DOE/EE-0767: Integrated Biorefineries: Biofuels, Biopower, and Bioproducts: www1.eere.energy.gov/biomass/pdfs/ibr_portfolio_overview.pdf). Currently, many of the co-products are diverted to low-value uses such as animal feed, anaerobic digestion, and nutrient recycling via fertilizers. However, in many biomass supplies the lipid components represent a higher-value, non-fuel product palette including nutritional supplements, feedstocks for bioplastics, and surfactants (IEA Bioenergy, “Bio-based Chemicals: Value-Added Products from Biorefineries”, 2012). The molecular components of the lipid fraction are typically triacylglycerides (TAGs), a non-polar fraction used as the precursor to biodiesel but also phospholipids, pigments, anti-oxidants, and sterols. Recent market data shows order-of-magnitude differences in the value of the various fatty acid fractions from TAG, the non-TAG lipids, and fuel or co-products (ICIS Pricing: 8 Aug. 2012, Fatty Acids—Fractionated (Asia Pacific): www.icispricing.com/il_shared/Samples/SubPage227.asp). In particular, the chain length and degree of unsaturation of individual fatty acids in TAGs strongly affects their value. In order to ensure the viability and sustainability of bio-based fuels, it is imperative to effectively take advantage of these differences in value in an integrated biorefinery.
Total biomass production via photosynthesis is estimated to be 200 billion tons/year, with less than 0.1% used by humans for non-nutritive purposes (Zoebelein, H., 2001, Dictionary of Renewable Resources. 2nd ed.; Wiley-VCH: Weinheim, Germany). Non-animal oils are found in highest concentrations in seeds (soybean, cotton), fruit pulps (palm, coconut), and various microalgae. The oil content varies widely and is rarely the major component of the raw biomass, which also contains protein and carbohydrate (Griffiths et al., 2011, J. Appl. Phycol. 24:989-1001; Liu, 1997, Soybeans: Chemistry, Technology, and Utilization. Chapman & Hall: New York). Depending on species and growth conditions, the oil fraction may contain a wide variety of nonpolar and polar lipids ranging from triacylglycerides, free fatty acids, phospholipids, antioxidants, pigments, vitamins, and sterols. Chain length and degree of unsaturation of the fatty acids and their derivatives are also highly variable. Separation and processing techniques that are robust and can tolerate varying biomass compositions will enable biorefineries to adapt to and efficiently use broader ranges of raw materials, and optimize for economic and environmental benefits.
The conventional approach to lipid extraction, processing, and refining involves pretreatment steps (pressing, wet or dry rendering), solvent extraction (e.g. hexane, chloroform/methanol), and often centrifuging to remove residue from water and oil phases. These methods are non-selective, yielding TAG as well as other lipid components including pigments, sterols, and phospholipids. Phospholipids may be separated by steam or chemical treatment, and free fatty acids removed by distillation or alkaline water washes (Zoebelein, H., 2001, Dictionary of Renewable Resources. 2nd ed.; Wiley-VCH: Weinheim, Germany). Further processing of TAG to isolate desired fractions is common. For example, processing of coconut fatty acids for use in surfactants requires additional energy-intensive steps: TAGs are hydrolyzed at high temperature and moderate pressure (250° C., 5 MPa) followed by vacuum distillation at 200° C. (Gervajio, 2012, Fatty Acids and Derivatives from Coconut Oil. In Kirk-Othmer Encyclopedia of Chemical Technology, Wiley). In the case of polyunsaturated fatty acids (PUFAs), which are sensitive to high temperatures, the isolated total lipids are typically converted to urea inclusion compounds and then undergo cryogenic fractional distillation at −20 to −70° C. (Mishra et al., 1993, Food Res. Int. 26:217-226) or chromatographic techniques involving AgNO3-modified solid phases, which are difficult to employ on a larger scale (Chester et al., 1996, Anal. Chem. 68:487R-514R).
In the production of biodiesel, extraction of lipids from biomass is followed by a stepwise transesterification reaction producing three fatty acid methyl ester (FAME) molecules for every molecule of TAG. Diglycerides (DGs) and monoglycerides (MGs) are produced as intermediates, with glycerol as a by-product of the overall reaction. The first step of the reaction (TAG to DG) is generally considered the rate-limiting step under ambient conditions, but the impact of solvent density has yet to be definitively determined (Lopez et al., 2007, J. Catal. 245:381-391). Since complete transesterification of one TAG molecule requires 3 molecules of alcohol, a minimal 3:1 molar ratio of methanol (or other alcohol) to substrate is required. To move from a second order to a pseudo-first order kinetics, excess methanol ratios are generally used (Fukuda et al., 2001, J. Biosci. Bioeng. 92:405-416). However, there is the potential for diminishing returns with excessive methanol ratios since high concentrations of methanol can adversely impact phase behavior in a mixed CO2 system (i.e., decreasing the solubility of TAG in the system).
In addition to an alcohol, the transesterification step typically requires an acidic, basic or enzymatic catalyst to carry out the reaction at temperatures below 100° C. Acidic catalysts can be used in feedstocks that may be contaminated with free fatty acids or water, preventing saponification that would occur with basic catalysts. However, basic catalysts have faster reaction kinetics than acidic catalysts (Fukuda et al., 2001, J. Biosci. Bioeng. 92:405-416). Lipases are also used for transesterification requiring a lower operating temperature, but are expensive compared to the alternatives (Fukuda et al., 2001, J. Biosci. Bioeng. 92:405-416), and reported yields under supercritical conditions are relatively low (Madras et al., 2004, Fuel 83:2029-2033; Taher et al., 2011, Biochem. Eng. J. 55:23-31).
There is a need in the art for energy-efficient methods of isolating biodiesel and other lipid-based products from biomass. The present invention addresses this unmet need.